Human body temperature increases during wakefulness and decreases during sleep. The body temperature rhythm (BTR) is a robust output of the circadian clock and is fundamental for maintaining homeostasis, such as generating metabolic energy and sleep, as well as entraining peripheral clocks in mammals. However, the mechanisms that regulate BTR are largely unknown. Therefore, there is a crucial need to identify the molecular mechanisms that regulate BTR. Drosophila are ectotherms, and their body temperatures are close to ambient temperature; therefore, flies select a preferred environmental temperature to set their body temperature. We identified a novel circadian output, the temperature preference rhythm (TPR), in which the preferred temperature in flies increases during the day and decreases at night. TPR thereby produces a daily body temperature rhythm. Fly TPR shares many features with mammalian BTR. During the current grant term, we established that Diuretic hormone 31 receptor (DH31R), a Drosophila calcitonin receptor family protein, mediates TPR, and we demonstrated that the closest mouse homolog of DH31R, calcitonin receptor (Calcr), is essential for normal BTR in mice. Importantly, both TPR and BTR are regulated in a distinct manner from locomotor activity rhythms, and neither DH31R nor Calcr regulate locomotor activity rhythms. Together, our findings suggest that DH31R/Calcr is an ancient and specific mediator of BTR. Thus, understanding fly TPR will provide fundamental insights into the molecular and neural mechanisms that control BTR in mammals. The goal of this proposal is to determine the molecular and neural mechanisms of TPR. Our recent study suggests that DH31 acts on clock neurons via DH31R to regulate TPR. Although DH31 primarily activates DH31R, DH31 can also activate the Pigment dispersing factor receptor (PDFR), required for locomotor activity rhythms, at a modest level in vitro. Because PDFR does not play a major role in daytime TPR, we expect that DH31R and PDFR are expressed in different cells. In addition to the identification of crucial ligand-receptor interactions, we recently found that master clock cells, the dorsal clock neurons 2 (DN2s), control TPR but not locomotor activity rhythms. The central hypothesis of this proposal: DN2s have temporally-regulated contacts with DN1ps and control rhythmic expression of DH31, which activates DH31R in PDFR-negative DN1ps, resulting in TPR. In Aim 1, we will elucidate the physical and functional relationship between DN1ps and DN2s to control TPR. In Aim 2, we will determine the mechanism that sets rhythmic DH31 expression in DN1ps. In Aim 3, we will determine the mechanism by which DH31R-expressing neurons control TPR. This project will contribute to a mechanistic understanding of fly TPR. The outcomes of this study should ultimately provide a novel mechanistic understanding of the mammalian BTR.